The present invention relates to the concentrated forms of TMB substrate in the field of immunoassays by using horseradish peroxidase (HRP) for the enzyme labeling of detection antibodies. Preferably, single component TMB substrate is used for the signal detection during various forms of immunoassays. The stability and concentrated forms of such TMB substrate become desirable during the large scale practical applications in immunodiagnostics to facilitate the transportation to distal or remote sites.